Procedure for designing efficient affinity cell separation processes

Claim 2

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells and separating the contacting surface and the media, wherein the improvement comprises the steps of: effecting relative movement between the media mixture of cells and the cell contacting surface at a nonsteady rate, thereby providing contact for a period of time between the cells and the contacting surface and increase the purity and yield of the separation; and maintaining a shear force at the contacting surface to a value less than developing bond strength between the target cells and the ligand.

Claim 3

The process set forth in claim 1 wherein the shear force divided by the square of the contact time is adjusted to between 2×10 -9 and 0.02 dynes/cm 2 sec 2 .

Claim 4

The process set forth in claim 1 wherein the shear force is adjusted to be less than the developing bond strength between the target cells and the contacting surface, said bond strength being proportional to the product of a rate factor and the square of the contact time.

Claim 5

The process set forth in claim 3 wherein the rate factor is adjusted by changing a variable selected from the group consisting of receptor density, receptor stability, receptor mobility, temperature, ligand affinity, ligand density, ligand mobility, ligand immobilization technique, media composition and cell-mediated behavior .

Claim 6

The process set forth in claim 4 wherein the cell contacting surface is provided by devices which permit cell contact at shear forces divided by the square of the contact time is between 2×10 -9 and 0.02 dynes/cm 2 /sec 2 and includes the step of adjusting the area of the contacting surface to be less than 10 cm 2 /ml of media.

Claim 7

The process set forth in claim 5 wherein the media is whole blood.

Claim 8

The process set forth in claim 1, which further includes adjusting media flow rate to obtain a target cell to contacting surface contact rate greater than that created by the inherent differential sedimentation velocity of the target cells and the contact surface.

Claim 9

The process set forth in claim 1, which further includes the step of adjusting the area of the contacting surface to be less than 10 cm 2 /ml of media.

Claim 10

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells and separating the contacting surface and the media, wherein the improvement comprises the steps of: effecting relative movement between the media mixture and the cell contacting surface for a contact time sufficient to attach the target cells to the contacting surface by an affinity bond between the cells and the surface; and maintaining a shear force at the contacting surface at a value less than developing bond strength between the target cells and the ligand.

Claim 11

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells and separating the contacting surface and the media, wherein the improvement comprises the steps of: effecting relative movement between the media mixture and the cell contacting surface for a contact time sufficient to attach the target cells to the contacting surface by an affinity bond between the cells and the surface; and maintaining a shear force at the contacting surface at a value less than developing bond strength between the target cells and the ligand.

Claim 12

The process set forth in claim 9 wherein the shear force divided by the square of the contact time is adjusted between 2×10 -9 and 0.02 dynes/cm 2 /sec 2 .

Claim 13

The process set forth in claim 9 or 10 which further includes the step of continuously renewing the cell contact surface with media containing the mixture of cells.

Claim 14

The process set forth in claim 10, which further includes the step of using one of a group consisting of rotating cylinders, flow inverters, thin film contactors, and Taylor-Couette devices to effect such surface renewal.

Claim 15

The process set forth in claim 10, which further includes the step of flowing the media mixture of cells against the cell contacting surface at a nonsteady flow rate, to increase the purity and yield of the separation.

Claim 16

The process set forth in claim 13, which further includes the step of adjusting the area of the contacting surface to be less than 10 cm 2 /ml of media.

Claim 17

The process set forth in claim 9, which further includes adjusting media flow rate to obtain a target cell to contacting surface rate greater than that created by the differential sedimentation velocity of the target cells and the contact surface.

Claim 18

The process set forth in claim 15 wherein the shear force divided by the square of the contact time is adjusted between 2×10 -9 and 0.02 dynes/cm 2 /sec 2 .

Claim 19

The process set forth in claim 9, which further includes maintaining the shear force at the contacting surface at a value intermediate of the developing bond strengths of the target cells and other cells that may be non-specifically bound to the contacting surface, to preferentially remove the non-specifically bound cells.

Claim 20

The process set forth in claim 9 wherein the shear force is increased at the end of the cell contacting process to preferentially remove non-specifically bound cells from the contacting surface.

Claim 21

The process set forth in claim 9 wherein the shear force is increased at the end of the cell contacting process to preferentially remove from the contacting surface cells of the media mixture that may be nonspecifically bound thereto.

Claim 22

The process set forth in claim 9 wherein the media is whole blood.

Claim 23

The process set forth in claim 20 wherein the media is diluted.

Claim 24

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells and separating the contacting surface and the media, wherein the improvement comprises the steps of: flowing the media mixture to contact the cell contacting surface to attach the target cells to the contacting surface by an affinity bond between the cells and surface; and maintaining the contact rate between the mixture cells and the contacting surface between 10 6 and 10 9 collisions/second/ml.

Claim 25

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells and separating the contacting surface and the media, wherein the improvement comprises the steps of: flowing the media mixture to contact the cell contacting surface to attach the target cells to the contacting surface by an affinity bond between the cells and surface; and maintaining the contact rate between the mixture cells and the contacting surface between 10 6 and 10 9 collisions/second/ml.

Claim 26

The process set forth in claim 22, which further includes the step of: maintaining a shear force at the contacting surface at a value less than the developing bond strength between the target cells and the ligand.

Claim 27

The process set forth in claim 23, which further includes the step of continuously renewing the cell contacting surface with media containing the mixture of cells.

Claim 28

The process set forth in claim 24 wherein the media is whole blood.

Claim 29

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells thereby providing specific binding sites for the target cells, but some of the cells in the mixture becoming non-specifically bound to the binding sites, and separating the contacting surface and the media, the improvement comprising the steps of: adjusting the surface area of the contacting surface to provide a concentration of cell binding sites which is less than 10 times the concentration of target cells in the media; maintaining a shear force at the contacting surface at a value less than the developing bond strength between the target cells and the ligand; and continuously renewing the cell contacting surface with media containing the cell mixture.

Claim 30

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells thereby providing specific binding sites for the target cells, but some of the cells in the mixture becoming non-specifically bound to the binding sites, and separating the contacting surface and the media, the improvement comprising the steps of: adjusting the surface area of the contacting surface to provide a concentration of cell binding sites which is less than 10 times the concentration of target cells in the media; maintaining a shear force at the contacting surface at a value less than the developing bond strength between the target cells and the ligand; and continuously renewing the cell contacting surface with media containing the cell mixture.

Claim 31

The process of claim 26, which further includes the step of: effecting non-steady movement between the cells and the surface.

Claim 32

The process set forth in claim 27 wherein the media is whole blood.

Claim 33

The process set forth in claim 26, which further includes adjusting media flow rate to obtain a target cell to contacting surface contact rate greater than that created by the inherent differential sedimentation velocity of the target cells and the contact surface.

Claim 34

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells thereby providing specific binding sites for the target cells, but some of the cells in the mixture becoming non-specifically bound to the binding sites, and separating the contacting surface and the media, wherein the improvement comprises the step of: adjusting the concentration of binding sites to a value less than or equal to the value calculated by setting the derivative with respect to S of the ratio of specific (B) to non-specific (N) cell binding equal to zero, where the ratio is defined by: ##EQU9## where S is the cell binding site concentration, T is the concentration of target cells, K is the equilibrium binding constant of the target cells and binding sites, T n is the concentration of cells in the media, and K n is equilibrium binding constant for cells non-specifically bound to the contacting surface; maintaining a shear force at the contacting surface at a value less than the developing bond strength between the target cells and the ligand; and effecting relative movement between the media mixture of cells and the cell contacting surface at a nonsteady rate, thereby to provide a contact for a period of time between the cells and the contacting surface and increase the purity and yield of the separation.

Claim 35

In a process for separating a target fraction of biological cells from a mixture of cells in a media by effecting contact between the cells in the mixture and a cell contacting surface having a ligand thereon with a specific affinity for the target cells thereby providing specific binding sites for the target cells, but some of the cells in the mixture becoming non-specifically bound to the binding sites, and separating the contacting surface and the media, wherein the improvement comprises the step of: adjusting the concentration of binding sites to a value less than or equal to the value calculated by setting the derivative with respect to S of the ratio of specific (B) to non-specific (N) cell binding equal to zero, where the ratio is defined by: ##EQU9## where S is the cell binding site concentration, T is the concentration of target cells, K is the equilibrium binding constant of the target cells and binding sites, T n is the concentration of cells in the media, and K n is equilibrium binding constant for cells non-specifically bound to the contacting surface; maintaining a shear force at the contacting surface at a value less than the developing bond strength between the target cells and the ligand; and effecting relative movement between the media mixture of cells and the cell contacting surface at a nonsteady rate, thereby to provide a contact for a period of time between the cells and the contacting surface and increase the purity and yield of the separation.

Claim 36

The process set forth in claim 30, which further includes the step of adjusting the area of the contacting surface to be less than 10 cm 2 /ml of media.